In the field of recombinant DNA technology it is often desirable to bind covalently a polynucleotide to other materials such as insoluble matrices, solid supports, proteins, small molecules or labels. For example, a polynucleotide bound to a detectable label can be used in procedures for detecting the presence of a particular polynucleotide in a biological sample. A polynucleotide bound to an insoluble matrix or a solid support or one bound to a small molecule capable to binding to such a matrix or support is useful for affinity separation and purification of a specific polynucleotide from a biological sample. The binding of the polynucleotide may be direct as, for example, by reaction between chemical moieties on the label or the surface of the support and moieties of the polynucleotide. In addition to direct covalent binding, the polynucleotide may be indirectly attached to the label or support by a linking or adapter molecule.
A general approach for covalent attachment of a polynucleotide to an insoluble matrix or solid support involves attachment directly to the activated matrix or support without the intervention of a adapter molecule or extensor arm. For example, DNA has been directly attached to cyanogen bromide (CNBr) activated matrices such as agarose. (Arndt-Jovin, D. J., Jovin, T. M., Bahr, W., Frischauf, A-M. and Marquardt, M. Eur. J. Biochem. (1975) 54, 411-418.). These workers assume that coupling of the polynucleotide to CNBr-activated agarose under the conditions used involves direct multipoint attachment of the aromatic groups of the bases. DNA has also been directly coupled with CNBr-activated Sephadex. (Siddell, S. G., Eur. J. Biochem. (1978) 92, 621-629.). Another example of direct coupling of DNA to activated cellulose is provided by Biagioni et al. These workers have attached DNA to dichlorotriazinyl cellulose, most likely through the amino groups of adenine, guanine and cytosine (Biagioni, S., Sisto, R., Ferraro, A., Caiafa, P. and Turano, C., Anal. Biochem. (1978) 89, 616-619.).
In addition to direct covalent linkage, a variety of methods have been reported for the indirect covalent attachment of a polynucleotide to a matrix or support by means of a bifunctional molecule first attached to the matrix or activated support. Noyes et al. report the indirect coupling of DNA to cellulose which has been diazotized. The resultant diazotized aryl amine of the cellulose reacts primarily with guanine and uracil (thymine) residues of single strands (Noyes, B. E., and Stark, G. R., Cell (1975) 5, 301-310.). See also Seed, B., Nucleic Acid Res. (1982) 10, 1799-1810. Another example of indirect, covalent attachment involves immobilization of polynucleotides to bisoxirane activated insoluble polysaccharides (Potuzak, H. and Dean, P. D. G., Nucleic Acids Res. (1978) 5, 297-303).
Alternatively, a polynucleotide may first be modified by a bifunctional molecule and subsequently attached to an insoluble matrix or solid support. For example, Dickerman et al. report derivatization of single-stranded DNA with 4-diazobenzoic acid and subsequent covalent attachment of the derivatized DNA to aminopentane Sepharose CL-4B (Dickerman, H. W., Ryan, T. J., Bass, A. I. and Chatterjee, N. K., Arch. Biochem. Biophys. (1978) 186, 218-234.). MacDougall, A. J., Brown, J. G., and Plumbridge, T. W., Biochem. J. (1980) 191, 855-858, describe the alkylation of double-stranded DNA with 4-bis (2-chloroethyl) amino-L-phenylalanine and immobilization of the resultant product of an insoluble support via the primary amino group of the phenylalanine moiety.
Each of the above methods of attaching a polynucleotide to a solid support, either directly or by first derivatizing the polynucleotide, has the significant disadvantage of reacting principally through the bases of the polynucleotide. The covalent linkages through the bases can be expected to interfere with the interactions of the immobilized polynucleotides with other macromolecules, particularly with hybridization reactions with other polynucleotides. This interference will be more severe for those hybridization reactions requiring a longer, uninterrupted region of free polynucleotide. Although the approach of the derivatizing a polynucleotide before subsequent immobilization allows greater control over the number of attachment points, covalent linkage is nonetheless through the bases of the polynucleotides.
Gilham has described direct attachment of polynucleotides to cellulose by a method involving specific activation of the terminal monosubstituted phosphate or polyphosphate of the polynucleotide by a water soluble carbodiimide. (Gilham, P. T., Biochemistry (1968) 7, 2809-2813.). The Gilham method is executed at pH 6 with an extremely large excess of the water soluble carbodiimide (present at greater than 200 mM), conditions under which the potential for base modification is increased. This potential for side reactions between carbodiimide and nucleotide bases or phosphodiester groups of the nucleotide backbone limits the utility of this approach. A similar scheme has been proposed by Urdea et al. (Urdea, M. S., Ghazi, H., Running J., Ku, L. and Warner, B. D., Poster Session; DNA and RNA Probes: Strategies and Applications: The Rensselaerville Institute Conference Center, Rensselaerville, N.Y.; Sep. 6-9, 1984.) in which 5' thiophosphate containing oligonucleotides are coupled with 2-bromoacetamide controlled pore glass. Although this method would be executed to provide some selectivity for the 5.dbd. end, attachment through the exocyclic amines is also possible. Moreover, low attachment yields are obtained.
Chu et al. have described a potentially simple method for attaching amines to the terminal 5'-phosphate of synthetic polynucleotides (Chu, B. C.. Wahl, G. M. and Orgel, L. E., Nucleic Acids Res. (1983) 11, 6513-6529.). The authors describe the coupling of uridine monophosphate to simple amines, polylysine and bovine serum albumin and the reaction of ethylene diamine with oligo(dT). The couplings are achieved by treating the 5'-phosphorylated species, either UMP or oligo(dT) with a water-soluble carbodiimide in imidazole buffer to form the corresponding 5'-phosphorimidazolide. The latter activated derivative is then isolated and treated with a large excess of amine to obtain a 5'-phosphoramidate. This chemistry requires a high concentration of amine to compete with water hydrolysis of the intermediate phosphorimidazolide. Because it is difficult to obtain relatively high concentrations of reactive amine groups on proteins and solid supports in an aqueous environment, very low levels of attachment, if any, will be achieved. If an immobilization takes place, a phosphoramidate bond is formed which is reported by Chu et al. to be unstable below pH 7.
van Boom et al. and Miyoshi et al. have described another immobilization chemistry for tethering 5'-spacer arm synthetic DNA. (Clerici, L., Campagnari, F., deRooij, J. F. M. and van Boom, J. H., Nucleic Acids Res. (1979) 6, 247-258; de Rooij, J. F. M., Wille-Hazeleger, G., Vink, A. B. J., and van Boom, J. H., Tetrahedron (1979) 35, 2913-2926; and (Miyoshi, K., Fuwa, T., European Patent Application, 0101985A1.). In this approach, synthetic DNA with an aliphatic amine spacer arm is reacted with a cyanogen bromide activated support, cellulose or agarose, and is reported to afford attachment only through the spacer arm's amine group. Although the coupling conditions are similar to those employed for immobilizing DNA through its exocyclic amines (see Arndt-Jovin et al., supra), Miyoshi and Fuwa argue that this form of attachment does not take place in their reaction scheme. They based their conclusions on the observation that DNA without the amine spacer arm did not show covalent linkage to activated cellulose or agarose. However, even though the initial attachment of DNA by their method is through the amine spacer arm, the likelihood of secondary attachment via the exocyclic amines of the nucleoside bases is increased once the DNA is attached and held in close proximity to the other activated sites on the support. Miyoshi and Fuwa report attachment of synthetic DNA per mg of support in the range of 0.1 to 0.3 nmole/mg.
A general method for covalently attaching DNA to enzymes has been disclosed by Renz. (Renz, M. and Kurz, C., Nucleic Acids Res. (1984) 12, 3435-3444.). The methods consists of first crosslinking the enzyme of interest to polyethyleneimine (PEI) with a crosslinking reagent, e.g., para-benzoquinone. This enzyme-PEI conjugate binds electrostatically to DNA via the PEI moiety and then can be covalently linked to single-stranded DNA with glutaraldehyde. It must be noted that this method is very cumbersome to execute, can be applied only to single-stranded DNA and, because attachment is through the DNA bases, would be expected to interfere with the hybridization reaction. In addition, cross linking an enzyme to PEI may significantly reduce its activity.
Several methods of covalent coupling of ferritin to the 3' terminus of RNA have been described. See Wu, M. and Davidson, N., J. Mol. Biol. (1973) 78, 1-21. In the simplest procedure a lysine amino group of the protein is coupled with the 3' terminal dialdehyde of the oxidized RNA and the resulting conjugate is stabilized by borohydride reduction. A second procedure modifies the protein with a --COCH.sub.2 Br group by acylation and attaches HSRCONHNH.sub.2 to the 3' oxidized RNA by hydrazone formation. The protein and RNA are then coupled by reaction of the --COCH.sub.2 Br and --SH groups. In a third and preferred procedure the 3' terminus of the RNA is modified with a --NHCH.sub.2 CH.sub.2 SH group and then coupled to the --COCH.sub.2 Br acylated protein. These procedures as described are applicable only to RNA because they require an oxidizable 2', 3' diol terminus. In addition, two of the three procedures require that the protein be acylated before attachment, a step which will destroy the activity of some enzymes.
Chemical and enzymatic methods have been used to attach several types of small molecules to DNA. Pyrimidine triphosphates modified at the C-5 position to contain biotin have been incorporated into DNA with various polymerases and terminal transferase. (Langer, P. R., Waldrop, A. A. and Ward. D. C., Proc. Natl. Acad. Sci. USA (1981) 78, 6633-6637; Leary, J. J., Brigati, D. J., and Ward, D. C., Proc. Natl. Acad. Sci. USA (1983) 80, 4045-4049; Murasugi, A. and Wallace, R. B., DNA (1984) 3, 269-277.). The C-5 position of pyrimidines has similarly been used to attach an EDTA derivative in a chemically synthesized oligodeoxynucleotide (Dreyer, G. B. and Dervan, P. B. Proc. Natl. Acad. Sci. USA (1985) 82, 968-972.). This form of substitution at pyrimidines is known to destabilize DNA hybrids (Langer et al., supra). The enzymatic methods are expensive, difficult to scale up, and sometimes lead to uneven levels of incorporation of biotinylated nucleotides.
The chemical method of Chu et al., supra has been extended to permit the attachment of biotin (via an activated biotinyl ester, Chollet, A. and Kawashima, E. H., Nucleic Acids Res. (1985) 13, 1529-1541) and an EDTA derivative (Chu, B. C. F. and Orgel, L. E. Proc. Natl. Acad. Sci. USA (1985) 82, 963-967.) to the 5' end of synthetic oligodeoxynucleotide. These attachments contain the same phosphoramidate linkage as the prototype method of Chu et al., supra, and therefore presumably suffer similar instability at pH's below neutrality.
Phosphotriester methodology has been used directly to attach a biotin derivative to the 5' end of a synthetic oligodeoxynucleotide (Kempe, T. Sundquist, W. I., Chow, F. and Hu, S.-L., Nucleic Acids Res. (1985) 13, 45-57.). The instability of the amide linkage joining the biotin to the oligonucleotide to the normal conditions of DNA deprotection drastically limit the length and sequence of biotinylated oligonucleotides prepared by this method. The longest biotinylated oligonucleotide chemically prepared by Kempe et al., was five nucleotides (50 is routine in present art) and contained no adenosine or guanosine residues (these require stronger conditions for deprotection, which would break the link to biotin). An intercalating dye (acridine) has been covalently attached to the 3' end of oligothymidylic acids by phosphotriester methods. See Asseline, U., Delarue, M., Lancelet, G., Toulme, F., Thuong, N. T., Montenay-Garestier, T. and Helene, C., Proc. Natl. Acad. Sci. USA (1984) 81, 3297-3301. These compounds were used to study acridine intercalation between adenine and thymine base pairs.
Although the above procedures will attach polynucleotides to other materials, most have the disadvantage of not being highly selective for the 5' or 3' end of a polynucleotide. These procedures can provide multipoint attachment and hence may not leave the bases of the polynucleotide available for successful hybridization reactions. In addition, many of these procedures provide low yields of attachment and employ reactions conditions that are known to modify polynucleotides. Furthermore, many of these methods yield linkages which are not stable over long periods of time or outside of a narrow pH range. Thus, alternative methods for attaching polynucleotides to other substances are being sought. It is highly desirable to have a method for attaching polynucleotides to other substances which is highly specific for attachment at the 5' or 3' end of the polynucleotide.